Western blot
Total proteins were extracted using RIPA buffer (0.5% Nonidet P-40, 0.1% sodium deoxycholate, 150mM NaCl, 50mM Tris-Cl, pH7.5 and 1x protease inhibitor cocktail). Cell lysates were subjected to high-speed centrifugation at 12000xg for 15min at 4°C. Protein concentrations were measured using Coomassie (Bradford) protein assay kit. Total soluble proteins were then separated on 10% SDS-PAGE and transferred into polyvinylidene difluoride membrane (Millipore, Billerica, MA). Membrane was blocked with 5% nonfat dry milk for 1h followed by incubation with diluted the primary antibodies (β-actin, 1:2000, Signalway antibody; Dip2a, 1:500, Novus; Dip2c, 1:1000, Abcam) for overnight at 4°C. Then the membrane was washed in TBST for three times, 5 min each and then incubated with secondary antibody (anti-rabbit horseradish peroxidase conjugate, 1:5,000; anti-mouse horseradish peroxidase conjugate, 1:5,000; Transgene) for 30min, followed by washing three times with TBST. Signals were detected using enhanced chemiluminescence Amersham™ ECL™ (GE Healthcare, USA) reagents. β-actin protein served as a loading control.